Figure 4

Accumulation of DNA:RNA hybrids in ICF mutant cells is a possible cause of DNA damage. (a) V5–RNASEH1_WT was transiently expressed in WT and ICF mutant cells; RNASEH1 was detected by anti-V5 antibody in green and DNA:RNA hybrids were detected by the S9.6 antibody in red (see Supplementary Fig. S6a). DNA:RNA hybrids positive cells in V5-RNASEH1_WT unexpressed (–) and expressed (+) cells were separately counted. Experiments were conducted in biological triplicate, and the total cell number examined (n) in more than ten images is shown in parentheses (each dot represents one image) (a–c). Data are mean ± s.e. (a–c). P values were obtained using the Mann–Whitney two-tailed U test (a–c). (b) V5–RNASEH1_WT was transiently expressed in WT and ICF mutant cells; RNASEH1 was detected by anti-V5 antibody in red and γH2AX foci were detected in green (see Supplementary Fig. S6b). γH2AX positive cells in V5-RNASEH1_WT unexpressed (–) and expressed (+) cells were separately counted. (c) V5–RNASEH1_WT was transiently expressed in WT and ICF mutant cells; RNASEH1 was detected by anti-V5 antibody in red and 53BP1 foci were detected in green (see Supplementary Fig. S6c). 53BP1 positive cells in V5-RNASEH1_WT unexpressed (–) and expressed (+) cells were separately counted. (d) Chromatin immunoprecipitation (ChIP) assay using anti-53BP1 antibody was conducted. WT and CDCA7 KO cells with or without transfection of the V5–RNASEH1_WT were served for the assay. Satellite-2 repeats, α-satellite repeats, two R-loop positive loci (TFPT and CALM3), and two negative loci, (EGR1 and SNRPN)³⁰, were amplified. Ct values of the two positive and negative loci were below the detection threshold. Amount of immunoprecipitated fragments was calculated as percent input. Experiments were conducted in biological duplicate and technical triplicate.